ABSTRACT
Toad urothelium barrier is the model to mimic and investigate urothelium permeability. Thiazide blocked ionic transportation in polarized membrane state. Jellyfish venom causes pores to be adjusted to urothelium permeability which improved polarization. This study aimed at CfTX-1 peptide in urothelium permeability evoked polarizations. Thiazide pretreated toad urothelium permeability to ions were investigated in modified Ussing chamber. Thiazide urothelium were further intervened by CfTX-1 peptide and treated with G-protein receptor agonists. 0.1 mol CaCl2 activated transurothelium potential differences were recorded by unipolar lead and computerized by fast Fourier transform technique. Apical chamber was settled as anode. The amplitude of potential differences were evaluated to determine the urothelium polarizations. The results indicated that CaCl2 activation induced a positive monophasic wave in thiazide urothelium, which suggested the urothelium was slightly polarized and significantly enhancive in adrenergic receptor treated urothelium. Furthermore, CfTX-1 peptide enhanced transurothelium potential difference in thiazide urothelium, therefore, urothelium were supra polarized. NPPB treatment significantly attenuated this supra polarization, which suggested that the Cl- influx was the main stream ionic compound of this polarization. It is concluded that CfTX-1 peptide was considered to generate supra polarization in thiazide urothelium. This mechanism is useful to study the improvement of drug delivery crossing urothelium barrier.
Key words: CfTX-1 partial sequence, toad urothelium, supra polarization, Cl- permeability.
Aurelia aurita nematocysts were dominated by large quantities of secretory proteolytic enzymes, displayed a wide spectrum of toxic activities that determined clinical relevance of the neurotoxicity, myotoxicity and hemolysis. Aurelia aurita crude venom was found to affect irreversible depolarization of the muscle membrane, increase in membrane permeability to sodium ions (Kihara et al., 1988), cell membrane lipid metabolism
(Helmholz et al., 2007), have a preferentially postsynaptic action at the neuromuscular junction by inhibiting acetylcholine binding to postsynaptic acetylcholine receptors (Ponce et al., 2013). Jellyfish venom, such as Carybdea rastoni, Chiropsalmus quadrigatus, contained diversified toxic isoforms were classified into at least two distinct subfamilies, type I and II. The box jellyfish Chironex fleckeri (C. fleckeri) toxin 1 and 2 were the short name of CfTX-1 and 2, two highly abundant toxins in the C. fleckeri venom, which was confirmed causing rapid cardiovascular collapse accompanied by hemolysis in anaesthetized rats (Yanagihara and Shohet, 2012). CfTX toxins shared high structural similarity with pore-forming three-domain Cry toxins produced by the bacterium Bacillus thuringiensis that possess the potent insecticidal activities (Podobnik and Anderluh, 2017)). The structural homology of Chironex fleckeri toxins and Cry toxins suggest that the toxins may have a similar pore-forming mechanism of action involving helices from the N-terminal domain (Brinkman, 2014), while structural variability within the toxin family may modulate receptor specificity. Therefore, CfTX-like toxins may be involved in oligomerization and pore formation not only on erythrocytes, but also other susceptible cells (Brinkman, 2007).
In isolated amphibian urinary bladder wall, mucosal and serosal surfaces have different permeability to solutes and water. Mucosal surface is normally impermeable to water (Peachey and Rasmussen, 1961; Parisi et al., 1981); however urothelium plasma membranes on the serosal surface are normally relatively permeable to circulation released stimulations (Michailova and Usunoff, 2006). Water permeability barrier is at mucous urothelium surface with striking properties of its polarity and the tight junctions (Parsons, 2007), while ionic traverse the barrier are involved in the ionic transporters located in apical membrane (Witten et al., 2018). The active transport in basal membrane induced variation of the permeability is evoked by Ca2+ and other solutes across the blood capillaries in serous. It has not been established whether one or the other of these layers is responsible for the unusual permeability characteristics of this cell surface. Cytological evidence indicated that the urothelium were structured by filamentous layer in the outside of the plasma membrane. That was the place to maintain the tight lateral attachment near the mucosal surface to assure an almost leak-proof bladder. Loosely interlocking folds of the lateral membranes probably help to have osmotic equilibrium with the serosal fluid crossing the urothelium barrier. Furthermore, serous as the urothelial sheet were more effect than mucous side in solutes transporting via the circulatory system into the urinary bladder sac. Ions either enter the cell through basal membrane surfaces or act at these surfaces lead to the permeability changes. The amplitude of transurothelium electrical potential difference is proportional to the ionic movement.
Based on the above previously knowledge, ionic permeability induced urothelium potentials in CaCl2 activation in basal membrane on serosal side was investigated. Moreover, the CaCl2 evoked transurothelium electrical potential difference in partial sequence of CfTX-1 pretreated urothelium preparations was analyzed.
Aurelia aurita nematocysts crude venom production
The crude venom were extracted from nematocyst of Aurelia aurita tentacle that obtained from Qiongzhou strait, Hainan, China. After homogenization nematocyst solution was centrifuged at 20,000×g for 1 h at 4°C. The proteins concentration in resultant supernatant was primarily determined by Bradford method, then immediately frozen at -80°C in condenser chamber and vacuum to extremely dry (VirTis BenchTop freeze dryer, SP industries, Inc., Pennsylvania, U. S. A.). Lyophilized concentrated crude venom was stored in -20°C for further analysis.
CfTX-1 peptide identification and synthesize
10% acrylamide gel was used to separate lyophilized concentrated crude venom by SDS-PAGE protocol. The target 43 kDa band cut off. The proteins were digested by trypsinase and the peptide fragments were entered into mass spectrometry analysis. The LC-MS/MS and peptide mapping method were used to identify CfTX-1 N-terminal fragment. Once the sequence of CfTX-1 was identified, this partial sequence of CfTX-1 were synthesized by using commercial resin solid-phase protocol and purified and final quality control by HPLC and electrospray Ionization Tandem Mass Spectrometry (ESI-MS).
Toad urothelium preparations
The toads were from the Hainan provincial drug safety evaluation center, Hainan province, China. The study protocol was approved in advance by ethics committee, Hainan Medical College. Toads were fed up in a humidity cage in the dark cycle prior to pithing. All toads were maintained under the surveillance of a veterinarian and adherence to policies and procedures from regulations for the administration of affairs concerning experimental animals.
The hemibladder wall were from double pithing toad, then prepared into approximately 9 mm x 9 mm in size, and balanced in isotonic amphibian Ringer's solution for 5 min to maintain the natural existence of urothelium osmotic gradient. The control preparation to the Thiazide pretreated was the urothelium only activated by CaCl2 but had no pretreatment and intervention; the control preparation in Thiazide pretreated was the urothelium only activated by CaCl2 but had no intervention by CfTX-1 peptide, acetylcholine, norepinephrine or 5-nitro-2-(3-phenylpropylamino) benzoic acid.
Thiazide (HCTZ) pretreated and CfTX-1 peptide intervened urothelium preparations were detected by using the hemibladder from the same toad urinary bladder. Tissue from at least four hemibladders with appropriate control preparations for comparison was subjected to each experimental condition that was studied. All experimental manipulations were performed within 2 h for avoiding the influences of membrane bioactivities.
Toad urothelium transurothelium potential difference analysis in mini Ussing chamber
The toad urothelium transurothelium potential differences measurement referenced Dunning-Davies method (Dunning-Davies et al., 2013). After rinsing with 0.9% saline solution, the intact urothelium layer were apical side down mounted on 80 mm2 circle surface on the modified mini Ussing chamber, where the dual chambers were vertically separated by horizontal mounted urothelium barrier to become apical chamber and basal chamber. The chambers were fulfilled with 0.9% saline solution and stabled in room temperature before investigations. For investigating the ionic crossing urothelium layer induced transurothelium potential difference (TPD), the preparations were initially activated urothelium basal membrane by 0.1 mol CaCl2 in basal chamber to obtain a primary TPD, then inspected the TPD in hydrochlorothiazide (HCTZ) pretreated urothelium preparations (HCTZ urothelium). The ionic crossing urothelium flowing induced TPD were measured by unipolar lead, and recorded with BL-420S biological signal acquisition system (Chengdu Taimeng Technology Ltd., Chengdu, China). The signals were computerized by fast Fourier transform technique with TM_WAVE software (Chengdu Techman Software Co. Ltd., Chengdu, China); apical side was settled as anode. Tissue from at least four hemibladders with appropriate control preparations for comparison was subjected to each experimental condition that was studied. All experimental manipulations were performed within 2 h to avoid the influences of membrane bioactivities (Kerec et al., 2005; Cohen et al., 2007).
CfTX-1 peptide evoked transurothelium potential difference in thiazide pretreated urothelium preparations
The HCTZ urothelium were further intervened by CfTX-1 peptide in basal chamber with dosage of 10 µg/ml. The muscarinic receptor agonist Acetylcholine (Ach, 2 mmol) or adrenergic receptor agonist norepinephrine (NE, 3 mmol) were treated to observe activating guanine nucleotide-binding proteins signaling induced permeability effect in CfTX-1 peptide intervened preparations. For understanding the ionic flow components that caused TPD in urothelium, 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 2mmol) were used to block the Cl- channel.
CfTX-1 identified in Aurelia aurita concentrated crude venom
In Figure 1a, lane 1 was Molecular PageRuler (Thermo Scientific, Inc Lithuania), lane 2, 3 and 4 were the reference bands of bovine serum albumin with the concentration of 10, 5 and 2.5 µg respectively. Lane 5, 6 and 7 were concentrated crude venom of 10, 5 and 2.5 mg, respectively. The compound in 43 kDa band of Figure 1a, lane 5 (the band with the arrow) was the target band for identifying sequence by LC-MS/MS. An 11 amino acid peptide sequence which had a high overlap with the positive strain of amino acid sequences 304 to 314 (IFNFFDLmKVK) of CfTX-1 N-terminal were confirmed (Figure 1b). This specific partial sequence was additionally synthesized by resin solid-phase synthesis. It was final purified to 99.75% (abbreviated as CfTX-1 peptide in the later). The quality control of peptide was monitored by HPLC and ESI-MS. Figure 1c was the 701.9Da peak value of purified CfTX-1 11 amino acid peptide product (Figure 1c).
CfTX-1 peptide in HCTZ urothelium preparations
In HCTZ urothelium, CaCl2 activation induced an increasing ionic current crossing the preparation, further generated a positive amplitude increasing of transurothelium potential difference (Figure 2a, 20.05±1.75 µV, n=5). Because HCTZ blocked the Na+/Cl- symporter in the apical membrane of urothelium, the remaining main ionic current were the ionic influx from the serosal basal side to the apical side (ionic influx). Furthermore, in CfTX-1 peptide intervened HCTZ urothelium, CaCl2 evoked significant increase in ionic influx current. This further induced secondary enhanced positive amplitude of transurothelium potential difference (Figure 2b, 120.74±23.66µV). This result suggested that CfTX-1 peptide significantly enhanced the ionic permeability in urothelium.
As the anode were settled in the apical of the urothelium in the Ussing chamber, the main stream of the ionic influx have been generated by the anion influx from the basal membrane.
NPPB attenuated transurothelium potential difference in thiazide pretreated and CfTX-1 peptide intervened preparation
NPPB was an inhibitor of many different Cl- channels and inhibit ATP release mediated by Cl- channels. It is an inhibitor of channel-mediated ATP release. In HCTZ pretreated and CfTX-1 peptide intervened preparations, NPPB significantly reduced CfTX-1 peptide induced ionic current influx and the amplitude of TPD (Figure 2c). The amplitude of TPD were attenuated to 26.67±8.62 µV, reduced 450% when compared with Figure 2b. NPPB attenuated TPD in Figure 2b suggested that CfTX-1 peptide triggered anion influx and TPD was Cl- influx.
CfTX-1 peptide intervened TPD attenuated in adrenergic receptor activated HCTZ urothelium
Figure 2d was the CaCl2 activated ionic permeability increasing induced amplitude of TPD in HCTZ urothelium that cholinergic receptor activated by Ach. In HCTZ urothelium, CaCl2 induced slightly but observable increased positive amplitude of potential difference. However, as shown in Figure 2f, HCTZ urothelium activated by adrenergic receptor agonist NE induced enhancement amplitude of TPD which suggested adrenergic receptors were the excitatory factor to increasing polarization in HCTZ urothelium. In HCTZ urothelium, Ach treated obtained 18.72±3.11ïV of TPD, moreover 82.40±9.55µV of TPD in NE treated HCTZ urothelium. A significantly results of CfTX-1 peptide intervene HCTZ urothelium was CaCl2 evoked TPD enhancement neither in Ach activated HCTZ urothelium nor in NE activated HCTZ urothelium (Figure 2e and 2g, marked with vertical arrows), which suggested the CfTX-1 peptide intervene evoked enhancement of urothelium polarization were inhibited by G-protein coupled receptors.
Summary of above results indicated that (1) CfTX-1 peptide intervene can evoke significantly urothelium polarization in HCTZ urothelium preparation. Furthermore, the main compound of ionic current induced supra polarization was Cl-, because NPPB can block this specific supra polarization. (2) CfTX-1 peptide evoked Cl- current was inhibited by G-protein coupled receptors.
In this study abundant of Aurelia aurita tentacles nematocyst proteins were identified from polyacrylamide gel. a specific sequence that belonged to a fragment of CfTX-1 N-terminal was identified. As the CfTX-1 N-terminal was function as the cellular outer membrane porin protein (OmpD), this study forward investigated this partial sequence of CfTX-1 evoking ionic permeability induced TPD in HCTZ urothelium. The results revealed that the CfTX-1 peptide represented enhanced positive peak potentials in anode on apical membrane, generated supra polarization on HCTZ urothelium. Moreover, the supra polarization was inhibited in adrenergic receptor activated HCTZ urothelium preparation. There is a precious reference report which suggested this result. Thurman and Higgins, 1988) reported that α2-adrenoceptor agonist was most potent for stimulating the amplitude of the short-circuit current in frog urothelium, and related to a simultaneous increase in the transepithelial flux of both chloride and sodium. Jellyfish proteins induced cell membranes pore formation was first reported in box jellyfish proteins (Nagai et al., 2000). Both CfTX-1 and CfTX-2 generated ring-shaped pores on cellular membranes which fold in two domains, larger N-terminal domain representing almost 75% of the structure, and a smaller C-terminal domain. Using tandem mass spectrometry, the proteome of Aurelia aurita crude venom profiled in 43 kDa band extracted peptide has significant sequence similarity to CfTX-1 N-terminal (Figure 1b). CfTX-1 which belongs to a family of pore-forming cnidarian toxins with N-terminal domain composed exclusively of a-helices and connecting loops have significant structural similarity to N-terminal domains of pore-forming 3d-Cry toxins (Brinkman, 2007, 2012). As the domain I of Cry toxin shares a significant structural similarity with the pore-formation domain of b-PFTs colicin A, CfTX-1 N-terminal estimated that might play an essential role in membrane penetration and pore formation after binding to the specific receptors.
Amphibian urinary bladder serosal surface was covered by an incomplete mesothelium, urothelium were water infiltrate in hypotonic in serous layer (Peachey, 1961). As is well known, Ca2+ applied directly to the mucosal surface has no effect, however, in the living toad the Ca2+ supply normally via the capillary network, which is on the serosal side of the epithelial sheet. Furthermore, since the mucosal surface of the bladder is normally impermeable to water, it does not seem that molecule as large as the ion could penetrate this barrier to express their activity. CaCl2 was used to activate serous chamber (basement membrane); this protocol reported increases [Ca2+]i through acting Na+/H+ transporter via basolateral membrane of the urothelium (Harvey and Ehrenfeld, 1988). Another cascade is the activation of phospholipase C and inositol 1,4,5-trisphosphate (IP3) pathway (Hanna-Mitchell and Gebruers, 2006). It is final release of Ca2+ from cytoplasmic storage. This induced secondary influx of Ca2+ via calcium release-activated chloride channels, as well as serous Ca2+ induced ATP increased [Ca2+]i in the absence of extracellular Ca2+ (Brodin et al., 1996; Brodin and Nielsen 2000). The thiazide urothelium generated dramatic enhanced positive potential by CfTX-1 peptide (Figure 2b), however, this enhancement were blocked by Cl- channel blockade NPPB (Figure 2c). It could be summarized that the CfTX-1 peptide significantly increased urothelium barrier permeability, increased Cl- influx on urothelium basal membrane. The role of chloride uptake is probably carrier mediated and driven by symporter or counter transporter. In thiazide urothelium chloride may pass through the pore formation of CfTX-1 peptide. Classic frog serous-urothelium membrane studies revealed that serous ATP activated in tight urothelium increased in [Ca2+]i, influenced both apical Na+ channels and basolateral K+ channels, activates chloride channels, plasma membrane proteins Ca2+ activated Cl− channels mediates the secretion of Cl−, bicarbonate and thiocyanate. In smooth muscle and excitable cells of the nervous system, calcium-dependent chloride channel (CaCCs) have an excitatory role coupling intracellular Ca2+ elevation to membrane depolarization, causes the appearance of Ca2+â€activated Cl− currents in native tissues (Ferrera et al., 2011). However, this classic mechanical is not enough to explain the extremely evoked urothelium hyperpolarization triggered by CfTX-1 peptide. Cellular membrane pore-forming, resulting in increasing membrane chloride conductance and, thus, depolarization of the urothelium was further dependent on membrane electrochemical gradient.
CfTX-1 peptide is a partial sequence of Chironex fleckeri toxins, which was identified from Aurelia aurita nematocysts from Qiongzhou Strait, Hainan province, China. The primary activation with CaCl2 indicated the increased permeability of anion accross thiazide urothelium, inducing secondary transurothelium potential enhancement. This supra polarization was probably induced by CfTX-1 peptide improved high permeability to Cl-. The Cl--induced supra polarization mechanism is meaningful in improving drug delivery crossing the urothelium barrier, and in promoting the drug clearance crossing the barrier.
CfTX-1, Chironex fleckeri Toxin 1; NPPB, 5-Nitro-2-(3-phenylpropylamino) benzoic acid; Ach, acetylcholine; NE, norepinephrine; HCTZ, hydrochlorothiazide.
China National University Students Innovation and Entrepreneurship Training Program (201711810020); Hainan Medical College Student Innovation and Entrepreneurship Training Program (HYCX2018081); Hainan Provincial Key Research and Development Project (ZDYF2017121).
The authors have not declared any conflict of interests.
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